1. Field of the Invention
The invention relates to procedures and devices for producing ions from a sample via interaction with metastable species.
2. Background of the Invention
Over the last decade, mass spectrometry has played an increasingly important role in the identification and characterization of biochemical compounds in research laboratories and various industries. The speed, specificity, and sensitivity of mass spectrometry make spectrometers especially attractive for rapid identification and characterization of biochemical compounds. Mass spectrometric configurations can be distinguished by the methods and techniques utilized for ionization and separation of the analyte molecules. One common method for ionizing biomolecules and organic compounds is electrospray ionization (ESI) whereby ions are ionized at atmospheric pressure outside the mass spectrometer via charging, dispersing and evaporating of small droplets. These ions are introduced into the vacuum of a mass spectrometer via an atmospheric pressure interface. Matrix-assisted laser desorption/ionization (ALDI) is another widely used method for ionization of larger biomolecules. In this technique, analytes are mixed with a matrix which absorbs laser irradiation and facilitates ionization. By using pulsed lasers for one-step desorption and ionization, MALDI has application under both reduced pressure and atmospheric pressure conditions.
Despite its extensive use in various applications, the mechanism of ion formation in the MALDI process has not yet been fully understood. It has been generally accepted that the matrix molecules go through a rapid phase change from the solid into the gas phase after absorption of laser radiation. The sublimated matrix molecules may form a dense multiphase gas plume embedding the analyte molecules. Ionization processes occurring during matrix-assisted laser desorption/ionization can be roughly divided into primary ionization in solid clusters and secondary ion-molecule charge- and proton-transfer reactions in the desorbed plume. Recent work (Karas et al., J Mass Spectrom. 35 (2000), the entire contents of which are incorporated herein by reference, suggests that primary ionization is the statistical occurrence of clusters with a deficit/excess of anions or cations. Highly charged positive ions cannot survive in the dense plume formed by the laser pulse as the ions undergo charge reduction to charge states 1 and 0, respectively, being neutralized in secondary reactions or in collisions with electrons. Electrons present in this process can be formed by a photoelectric effect on the metal/organic matrix interface, as described in Frankevich et al., Int. J. Mass Spectrom. 220, 11 (2002), the entire contents of which are incorporated herein by reference.
Hence, neutralization can be a prominent process and the singly-charged ions finally observed may be considered the “lucky survivors.” Experimental measurements of the ion to neutral ratio formed in MALDI process has been reported as low as 10−4-10−7. As a result, more than 99.99% of analyte molecules are present in the gas phase as neutrals and therefore would not contribute to the ion signal.
Franzen et al., U.S. Pat. No. 5,663,561, the entire contents of which are incorporated herein by reference) address a low ionization of the MALDI process by using a laser to desorb the matrix/sample mixture in an atmospheric pressure region and thereafter separate reagent ions from a corona discharge to subsequently chemically ionize neutral sample molecules. Coon et al., U.S. Pat. No. 6,838,663, the entire contents of which are incorporated herein by reference, describe desorbing neutral molecules by laser irradiation from a wide group of supporting structures including: polyacrylamide gel, a thin-layer chromatography plate, a biological tissue, an agarose gel, paper, a fabric, a polymer, plastic, geological material, soil, biological solution, blood plasma and others. The reagent ions were described therein as being generated by corona discharge and mixed with neutral sample molecules at atmospheric pressure. Thomson et al., U.S. Patent Application 2003/0111600 A1, the entire contents of which are incorporated herein by reference, describe vaporization of sample molecules and mixing of the vaporized molecules into a corona discharge to generate ions at sub-atmospheric pressure. The drawback of such arrangements is the necessity of creating a corona discharge of large concentrations of reagent ions. These ions from the corona discharge can charge the surfaces of atmospheric pressure interfaces and ion optics, thus reducing the transmission of analyte ions, usually present in small quantities.
An atmospheric pressure ionization source using metastable atom bombardment is described in Cody, et al., Anal. Chem. 2005, 77: 2297-2302, the entire contents of which are incorporated herein by reference. Cody et al., U.S. Pat. Appl. Publ. No. 2005/0056775, the entire contents of which are incorporated herein by reference, provide further details of an atmospheric pressure ionization source using metastable atom bombardment. Ionization of small inorganic molecules at reduced pressures was described for example, in Bertrand et al. U.S. Pat. No. 6,124,675, the entire contents of which are incorporated herein by reference, and in Lewis, et al., Anal. Chem. 2003, 75: 1983-1996, the entire contents of which are incorporated herein by reference. As disclosed in U.S. Pat. No. 6,124,675, a beam of metastable atoms can be generated from a source of rare gas. The rare gas is typically introduced into a chamber having a pressure gradient from its entry to an exit. By applying electrical energy to a cathode and anode, an electric discharge can be generated between the cathode and the anode, thereby extending through the aperture or nozzle into the chamber. The discharge in turn energizes the atoms of the rare gas into a mixture of ions/electrons and metastable atoms in which the electrons of these atoms can be raised to higher energy levels. The stream of metastable atoms, ionized atoms and electrons can then pass through a charged deflector, which removes some of the ions/electrons from the stream of particles. Since the metastable atoms are not charged, the forces applied on the ions and electrons tend to force these particles towards a longitudinal axis extending between the cathode and anode while metastable species are not affected.
In these techniques, ionization of small inorganic molecules in the gas phase has been accomplished by the use of metastable atom bombardment, in which a neutral metastable species is used to bombard the sample molecules. A reaction system (which produces a beam of metastable atoms) includes a reaction vessel having a source of rare gas at one end of the vessel, a cathode positioned inside the vessel, and a small sonic nozzle placed at the other end of the vessel. Outside the vessel, a generally cone shaped anode (referred to as a “skimmer”) includes an aperture at the apex of the cone. Behind the skimmer, a set of plates serves as a deflector. In operation, the gas is detected at one end of the vessel and passes through the nozzle at the opposite end. The atoms of gas, which are injected through the discharge, are energized to a metastable state, with some of the gas atoms being energized to the point of ionization, thus releasing free ions and electrons into the metastable gas stream. The metastable gas, the free ions and electrons then pass through the aperture in the apex of the skimmer into a set of charged deflector plates. Free ions/electrons are attracted to the deflector plates, leaving the relatively charge free, metastable gas particles to pass through the deflector plates and bombard the sample molecules.